In a sequential staining like this, the most worry comes from the 2nd secondary antibody (594 here) binding to the 1st primary antibody (anti-alpha here). So the fixing in the middle is a measure to prevent this.

The result of this expt was as follow:
(1) No signals in 488 and 594
(2) No signals in 488 and 594
(3) Signals in 488, but not 594
(4) Signals in 488 and 594

I was expecting signals in 594 for condition 2, but I did not get it. I thought it was a technical error, but the same result was repeated 3 times. Since there's no 594 signals in condition 3, there should not be any significant binding of anti-rb 594 to anti-alpha. So that would mean the 594 signal in condition 4 is real. But without anti-alpha, I did not get the 594 signal in condition 2. So I'm very puzzled. Can someone please help? Thanks!

-alexkoon-

Have you tried doing the 594 ab first rather than the 488?

-bob1-

Haven't tried that yet... I am about to do it this weekend or next week.

Btw, now that I have taken images of everything, there IS actually some staining of 594 on condition 2. But it's extremely weak comparing to condition 4. If the intensity of 594 in condition 4 is 10, intensity of 594 in condition 2 is only 4. Normally, this indicates that the anti-rb 594 is binding to anti-alpha. But comparing condition 3 with condition 4 indicates that such binding is very little.